Difference between revisions of "Team:Nanjing-China/Notebook"
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<div class="word-1" id="April" style="display:none; overflow:hidden;"> | <div class="word-1" id="April" style="display:none; overflow:hidden;"> | ||
<div class="bottom-2">4</div> | <div class="bottom-2">4</div> | ||
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<h4>Human Practices,Collaboration&Society:</h4> | <h4>Human Practices,Collaboration&Society:</h4> | ||
<p>We learned about nitrogenous fertilizer production status in quo and the advancement of China’s nitrogenous fertilizer industry. In order to better understand the actual demand of nitrogenous fertilizer in agriculture, we decided to visit farmers in Xiaohe Bei Village.<br /> | <p>We learned about nitrogenous fertilizer production status in quo and the advancement of China’s nitrogenous fertilizer industry. In order to better understand the actual demand of nitrogenous fertilizer in agriculture, we decided to visit farmers in Xiaohe Bei Village.<br /> | ||
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We held conferences with Nanjing Agricultural University and China Pharmaceutical University to share experiences of being IGEMers. </p> | We held conferences with Nanjing Agricultural University and China Pharmaceutical University to share experiences of being IGEMers. </p> | ||
</div></div> | </div></div> | ||
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<h4>Technical works Wet&Dry labs:</h4> | <h4>Technical works Wet&Dry labs:</h4> | ||
<p>Wet Lab:Having confirmed the theme of our project, we began to work on our design. We read latest papers about biological nitrogen fixation, focused on the method sections and discussed what we didn’t understand in details. During the last week of this month, we worked out the first version of our design.<br /> | <p>Wet Lab:Having confirmed the theme of our project, we began to work on our design. We read latest papers about biological nitrogen fixation, focused on the method sections and discussed what we didn’t understand in details. During the last week of this month, we worked out the first version of our design.<br /> | ||
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<div class="word-1" id="May" style="display:none; overflow:hidden;"> | <div class="word-1" id="May" style="display:none; overflow:hidden;"> | ||
<div class="bottom-2">5</div> | <div class="bottom-2">5</div> | ||
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<h4>Human Practices,Collaboration&Society:</h4> | <h4>Human Practices,Collaboration&Society:</h4> | ||
<p>We planned to investigate the current production of nitrogenous fertilizer so we prepared interview questions and contacted Yantai Wuzhou Feng Fertilizer Plant. Then we went there, met the manager and were shown around the factories. We communicated with the technical R&D personnel and realized the big challenge we had to overcome before putting our project into practical application.</p> | <p>We planned to investigate the current production of nitrogenous fertilizer so we prepared interview questions and contacted Yantai Wuzhou Feng Fertilizer Plant. Then we went there, met the manager and were shown around the factories. We communicated with the technical R&D personnel and realized the big challenge we had to overcome before putting our project into practical application.</p> | ||
</div></div> | </div></div> | ||
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<h4>Technical works Wet&Dry labs:</h4> | <h4>Technical works Wet&Dry labs:</h4> | ||
<p>Wet lab:We measured the transcriptional activity of nif promoter and constructed the complete line of nif cluster, BBa_K1796015. Then we transformed the plasmid Pcb1C3 containing the nif cluster and the fusion protein expression plasmid including <em>E. coli</em> outer membrane protein OmpA and the PbrR protein into <em>E. coli</em> strain pUC57. Besides,based on our Human Practice, we modified our design by adding Cd2+ toxicity test to it.</p> | <p>Wet lab:We measured the transcriptional activity of nif promoter and constructed the complete line of nif cluster, BBa_K1796015. Then we transformed the plasmid Pcb1C3 containing the nif cluster and the fusion protein expression plasmid including <em>E. coli</em> outer membrane protein OmpA and the PbrR protein into <em>E. coli</em> strain pUC57. Besides,based on our Human Practice, we modified our design by adding Cd2+ toxicity test to it.</p> | ||
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<div class="word-1" id="July" style="display:none; overflow:hidden;"> | <div class="word-1" id="July" style="display:none; overflow:hidden;"> | ||
<div class="bottom-2">7</div> | <div class="bottom-2">7</div> | ||
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<div style=" padding:20px;"> | <div style=" padding:20px;"> | ||
<h4>Human Practices,Collaboration&Society:</h4> | <h4>Human Practices,Collaboration&Society:</h4> | ||
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We were interviewed by Nanjing University Student Career Guidance Center. The Wechat Push introducing our team was issued on the public account “NJU Employment”. </p> | We were interviewed by Nanjing University Student Career Guidance Center. The Wechat Push introducing our team was issued on the public account “NJU Employment”. </p> | ||
</div></div> | </div></div> | ||
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<h4>>Technical works Wet&Dry labs:</h4> | <h4>>Technical works Wet&Dry labs:</h4> | ||
<p>Wet lab:We finished the last step in construction of whole-cell photocatalytic nitrogen fixation system----biosynthesis of CdS semiconductor. Then we performed gas chromatography to detect the amount of acetylene reduced to indirectly test the nitrogen fixation activity of our system. <br /> | <p>Wet lab:We finished the last step in construction of whole-cell photocatalytic nitrogen fixation system----biosynthesis of CdS semiconductor. Then we performed gas chromatography to detect the amount of acetylene reduced to indirectly test the nitrogen fixation activity of our system. <br /> | ||
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<div class="word-1" id="August" style="display:none; overflow:hidden;"> | <div class="word-1" id="August" style="display:none; overflow:hidden;"> | ||
<div class="bottom-2">8</div> | <div class="bottom-2">8</div> | ||
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<h4>Human Practices,Collaboration&Society:</h4> | <h4>Human Practices,Collaboration&Society:</h4> | ||
<p>We distributed brochures about our project at NJU.<br /> | <p>We distributed brochures about our project at NJU.<br /> | ||
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We helped Central South University to found team.</p> | We helped Central South University to found team.</p> | ||
</div></div> | </div></div> | ||
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<h4>Technical works Wet&Dry labs:</h4> | <h4>Technical works Wet&Dry labs:</h4> | ||
<p>Wet lab:We conducted ICP-MS measurement of Cd2+ adsorption, Cd2+ toxicity test and TEM-EDX analysis. In addition,we improved our part to make it easier to operate.<strong> </strong><br /> | <p>Wet lab:We conducted ICP-MS measurement of Cd2+ adsorption, Cd2+ toxicity test and TEM-EDX analysis. In addition,we improved our part to make it easier to operate.<strong> </strong><br /> | ||
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<div class="word-1" id="September" style="display:none; overflow:hidden;"> | <div class="word-1" id="September" style="display:none; overflow:hidden;"> | ||
<div class="bottom-2">9</div> | <div class="bottom-2">9</div> | ||
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<h4>Human Practices,Collaboration&Society:</h4> | <h4>Human Practices,Collaboration&Society:</h4> | ||
<p>We performed Language project with IIT Madras and received the finished video a few days later. <br /> | <p>We performed Language project with IIT Madras and received the finished video a few days later. <br /> | ||
We helped another IGEM team, CSU-China to establish their team. We issued our Emoji chanllenge on the official website and received some interesting feedback shortly after that.</p> | We helped another IGEM team, CSU-China to establish their team. We issued our Emoji chanllenge on the official website and received some interesting feedback shortly after that.</p> | ||
</div></div> | </div></div> | ||
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<h4>Technical works Wet&Dry labs:</h4> | <h4>Technical works Wet&Dry labs:</h4> | ||
<p>Hardware: Inspired by our Human Practice, we formed an idea of designing a device for the growth of engineered E.coli strain<em>. </em>First we drew a draft on paper and then used the software solidworks to draw a 3D version draft. Eventually, a real device came out. The device provided a great help to our further experiments.</p> | <p>Hardware: Inspired by our Human Practice, we formed an idea of designing a device for the growth of engineered E.coli strain<em>. </em>First we drew a draft on paper and then used the software solidworks to draw a 3D version draft. Eventually, a real device came out. The device provided a great help to our further experiments.</p> | ||
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The thick carbon film (20 to 30 nm) on the copper grid was immersed in the bacteria solution for 1 second before imaging, dried under atmospheric conditions, and then imaged using TEM. At the same time, the EDX system (EDAX, AMETEK) was attached to the microscope for elemental analysis. All TEM images were imaged using a JEOL JEM-2100 electron microscope at an acceleration bias of 200 kV. </p> | The thick carbon film (20 to 30 nm) on the copper grid was immersed in the bacteria solution for 1 second before imaging, dried under atmospheric conditions, and then imaged using TEM. At the same time, the EDX system (EDAX, AMETEK) was attached to the microscope for elemental analysis. All TEM images were imaged using a JEOL JEM-2100 electron microscope at an acceleration bias of 200 kV. </p> | ||
</div> | </div> | ||
| − | <div class="word" id="reference"> | + | <div class="word" align="left" id="reference"> |
<h2>Reference</h2> | <h2>Reference</h2> | ||
<ol> | <ol> | ||
Revision as of 08:01, 14 October 2018
March
April
MAY
June
July
August
September
Protocol
ICP-MS(Inductively Coupled Plasma Mass Spectrometry) measurement of Cd2+ adsorption
Escherichia coli BL21 containing OmpA-PbrR-PJQ200SK (pBAD33) plasmid was cultured in LB medium to an OD600 of 0.4-0.6. Arabinose and CdCl2 were added to the medium to a final arabinose concentration of 40 μM and a final Cd2+ concentration of 100 μM, to induce the formation of CdS nano semiconductors.
From the start of the induction, 5 ml of the bacterial solution was taken from the culture every 6 hours (sampling to 24 hours), centrifuged at 4000 rpm for 2 minutes, and washed three times with water to remove the medium involved in the bacterial surface.
The washed bacteria were resuspended in 5 ml of water. OD600 was measured, and the bacteria were collected by centrifugation.
3 ml of concentrated nitric acid was added and the mixture was digested overnight at 90 °C.
The Cd2+ content in the sample was measured using ICP-MS.
Cd2+ toxicity test
Multiple groups of LB medium were prepared, and arabinose with a final concentration of 40 μM and different amounts of CdCl2 were added to the medium to form a Cd2+ gradient of 0,150 μM, 300 μM, 600 μM, and 1000 μM.
E. coli BL21 containing the OmpA-PbrR-PJQ200SK (pBAD33) plasmid and plasmid-free E. coli BL21 (control) were cultured in different media.
The OD600 value was measured every 2 hours and measured for 12 hours.
Transmission electron microscopy with energy-dispersive x-ray spectroscopy (TEM-EDX)
After the Cd2+ adsorption induction was completed, the bacteria were collected by centrifugation and resuspended in ultrapure water. Samples were sent for TEM image acquisition.
The thick carbon film (20 to 30 nm) on the copper grid was immersed in the bacteria solution for 1 second before imaging, dried under atmospheric conditions, and then imaged using TEM. At the same time, the EDX system (EDAX, AMETEK) was attached to the microscope for elemental analysis. All TEM images were imaged using a JEOL JEM-2100 electron microscope at an acceleration bias of 200 kV.
Reference
- Kathryn RF,Yanning Z,et.al.(2016)Light-driven carbon dioxide reduction to methane by nitrogenase in a photosynthetic bacterium PNAS
- Wang L,Zhang L,Liu Z,Zhao D,Liu X et.al(2013) A minimal Nitrogen Expression of Active Nitrogenase in Escherichia coli PLOS Genetics9(10):e1003865
- Katherine AB,Derek FH,Molly BW et.al(2016) Light-driven nitrogen reduction catalyzed by a CdS:nitrogenase MoFe protein biohybrid Science352,448
- Wei W,Sun PQ,Li Z,Song KS,Su WY,Wang B,Liu YZ,Zhao J et.al (2018) A surface display biohybrid approach to light-driven hydrogen production in air Science eaap9253
- Wei W,Zhu T,Wang Y et.al(2012) Engineering a gold-specific regulon for cell-based visual detection and recovery of gold Chem.Sci,3,1780-1784
- James BH&Douglas CR(1996) Structural Basis of Biological Nitrogen Fixation Chem.Rev.96,2965-2982
Address
Life Science Department
#163 Xianlin Blvd, Qixia District
Nanjing University
Nanjing, Jiangsu Province
P.R. of China
Zip: 210046
